Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
Passage from G1-into S-phase of the eukaryotic cell cycle is dependent on the successive activation and inactivation of several G1-cyclin-dependent protein kinases (G1-Cdks). These G1-Cdks function to coordinate transcription control with the activity of numerous cell cycle regulatory proteins and genes, whose products mediate DNA synthesis and other mechanical aspects of the cell cycle (Dynlacht 1997). During normal cell cycle progression, the temporal activation of individual G1-Cdks is dictated primarily by the expression of cognate cyclins together with both activating and inactivating phosphorylation. In addition G1-Cdk activity is also affected by the interaction with regulatory proteins, such as p21WAF1/Cip 1, which inhibit Cdks as part of a complex cellular response to genotoxic stress. This additional layer of regulation affords cells with damaged DNA the ability to interrupt or delay cell cycle progression at “G1- and G2-cell cycle checkpoints” (Peter and Herskowitz 1994; Elledge and Harper 1994; Hunter and Pines 1994).
The p21WAF1/Cip 1 gene, also known as pic1, Sdil or 20CAP (Duttaroy et al. 1997; Gu et al. 1993), encodes an inhibitor of most cyclin-dependent kinases (Xiong et al. 1993; Harper et al. 1995) and has been implicated as a growth arrest mediator in p53-tumor suppression. In response to DNA damage (Macleod et al. 1995; Gottlieb and Oren 1996) or alterations in cellular homeostasis (Chernova et al. 1995), normal cells can respond by increasing the induction of p53. Although the mechanism(s) leading to the induction of p53 are unclear, p53 acts as a transcription factor, and increased expression of wild-type p53 stimulates the synthesis of p21WAF1/Cip 1. In turn, p21WAF1/Cip 1 inhibits the activity of cyclin D/Cdk4,6 and/or cyclinE/Cdk2, preventing the phosphorylation of the retinoblastoma protein (Rb) and inducing cell growth arrest late in the G1 stage of the cell cycle (Chernova et al. 1995). Studies in p53 deficient cells (Agarwal et al. 1995; Yin et al. 1992) indicate that the increased expression of p53 alone (i.e. in the absence of DNA damage) stimulates the synthesis of p21WAF1/Cip 1 and growth arrest in the G1 phase of the cell cycle. Nonetheless, recent studies indicate that p21WAF1/Cip 1 is also required for arrest of the cell cycle by the tumor-suppressor protein BRCA1, which is associated with hereditary breast and ovarian cancer (Somasundaram et al. 1997; Scully et al. 1997), and p21WAF1/Cip 1 induction has been implicated in other physiological processes, such as cell differentiation and senescence, via p53 independent mechanisms (Zeng and el-Deiry 1996).
In vivo, the reversible phosphorylation of serine residues is believed to influence the biological activity of p53, and p53 is phosphorylated at multiple sites (Takenaka et al. 1995; Hecker et al. 1996; Mayr et al. 1995). Increased phosphorylation of ser 309 and 370 enhances the DNA binding activity of p53, and phosphorylation of serines 6, 17 and 34 is important for transcriptional activity (Takenaka et al. 1995; Hecker et al. 1996; Mayr et al. 1995).
Since virtually all known examples of transcriptional control during the cell cycle involve phosphorylation and the G1-Cdks are serine/threonine (ser/thr) protein kinases, specific ser/thr protein phosphatases (PPases) may also participate in the regulation of cell cycle progression. In mammals, at least thirteen closely related enzymes, including four highly homologous isoforms of PP1 (PP1α, β, γ1, γ2), two isoforms of PP2A (PP2α,β), three isoforms of PP2B (α,β,γ), PP4, PP5, PP6 and PP7 have been identified, and any, or all, of these PPases may contribute to cell cycle regulation (Cohen 1997; Egloff et al. 1995; Brewis et al. 1993; Chen et al. 1994; Bastians and Ponstingl 1996). Of the known PPases, PP1, PP2A, PP4 and PP5 are sensitive to several natural toxins, such as okadaic acid (Bialojan and Takai 1988; Honkanen et al. 1994; Cohen et al. 1990b) and microcystin (Honkanen et al. 1990), and reports indicating that okadaic acid has tumor promoting activity have led to speculation that the toxin-sensitive PPases may regulate cell growth. In addition, preliminary studies indicate that tumor cells in log phase growth express higher levels of PP5 mRNA, and the presence of multiple tetratrico peptide repeat (TPR) sequences in PP5 suggest that PP5 may interact with other TPR containing proteins, many of which are involved in the regulation of cell cycle progression (Lamb et al. 1995).
Determining specific roles for any of the thirteen structurally related mammalian PPases has proven difficult due to: 1) the lack of substrate sensitivity demonstrated by most of these PPases in vitro; 2) the lack of truly specific inhibitors for individual PPases; and 3) complications in assessing the functions of numerous loosely associated ancillary proteins that regulate the activity of, impart substrate specificity to, and influence the cellular localization of individual PPases, in vivo (Walter and Mumby 1993; Shenolikar and Nairn 1991; Cohen 1989; Cohen et al. 1990a).
Since a predisposition to many forms of cancer has been attributed to defects in tumor suppressor genes that regulate the expression of the cyclin-dependent kinase inhibitor, p21WAF1/Cip 1, any methods that can circumvent these defects could potentially be useful in treating and/or preventing cancer.